The role of Eps8/Src axis in macrophage-mediated innate immunity

Abstract

We earlier demonstrated iNOS/Src/FAK axis as a general mechanism of macrophage motility in response to various pathogen-associated molecular patterns (PAMPs) and Eps8 took part in TLR4-mediated signal transduction enhancing phagocytosis and bacterial killing effect In the first study we found that dsRNA stimulation induces biphasic TLR3 Tyr-759 phosphorylation in macrophages In addition to the immediate TLR3 Tyr-759 phosphorylation we identified a second wave of Tyr-759 phosphorylation accompanied by an increase of both Src and ifn-β transcription in the later phase of dsRNA stimulation Interestingly Src phosphorylated TLR3 Tyr-759 in vitro and in vivo However knockdown of Src abolished the late phase of TLR3 Tyr-759 phosphorylation and decreased the nuclear accumulation of interferon regulatory factors 3 and 7 (IRF3 and -7) and IFN-β production Reintroduction of Src restored all of these molecular changes Notably via down-regulation of Src dsRNA-elicited TLR3 Tyr-759 phosphorylation the nuclear accumulation of IRF3/IRF7 and IFN-β generation were inhibited in inducible nitric-oxide synthase (iNOS)-null macrophages TLR3 knockdown destabilized Src and reduced the nuclear level of IRF3/IRF7 and IFN-β production in macrophages exposed to LPS (a TLR4 ligand known to induce Src and IFN-β expression) Ectopic expression of wild type TLR3 but not its 759-phenylalanine mutant restored Src activity and ifn-β transcription Taken together these results suggested an essential role of the iNOS/Src/ TLR3 axis in IFN-β production in macrophages In the second study we observed that expression of Eps8 was PAMP-inducible and iNOS/Src-dependent Attenuation of Eps8 simultaneously impaired Src activity and suppressed macrophage mobility Notably ectopic Eps8 partly restored motility and Src activity in Src-attenuated macrophages exposed to PAMPs These findings indicated Eps8 modulating TLR4-mediated signal transduction and taking part in Src-mediated cell migration in TLRs-stimulated macrophages In the third study we found that Src knockdown impaired LPS-induced NO production and cytokines secretion which was reverted by ectopically expressed avian Src Attenuation of Eps8 also reduced LPS-mediated iNOS expression and Src activation Indeed via activation of NF-κB signaling Eps8 affected NO production and the secretion of TNF-? IL-1β and IL-6 Taken together our data indicates that Eps8 and Src are necessary for LPS mediated NF-κB activation and contributes to macrophage-mediated innate immunity

Date of Award	2015 Jul 8
Original language	English
Supervisor	Tzeng-Horng Leu (Supervisor)